| Liver cirrhosis is a common pathological process,which often secondary to liver inflammation or fatty liver and other liver diseases.In the theory of Traditional Chinese Medicine(TCM),it belongs to the category of hypochondriac pain,jaundice,tympanites and so on.Current clinical studies suggest that TCM has special curative effect for liver cirrohsis.According to the theory of TCM,Lycium Barbarum could nourishing liver and kidney,and thus was used in the treatment of liver cirrhosis for some syndrome types in clinic.Betaine,one of the main components of Lycium Barbarum,contains methyl,which is involved in the methylation regulation of some genes.It is generally believed that DNA methylation may lead to chromosome condensation and inhibit gene expression.At the same times,study shows that epigenetic regulation plays an important role in the development of liver fibrosis.Therefore,we carried out a meta-analysis of the anti-cirrhosis effect of Lycium Barbarum,studied the changes of DNA methylation level in liver cirrhosis,and discussed whether betaine can improve liver fibrosis by inhibiting DNA methylation of these related genes.Part Ⅰ Function of Lycium Barbarum contained Traditional Chinese Medicine in the treatment of liver cirrhosis:a meta-analysisObjectives:to explore the application of Lycium Barbarum contained Traditional Chinese Medicine in the treatment of liver cirrhosis and analyze its effect,thus provide instructions for clinical while use of Lycium Barbarum.Methods:firstly,four databases include SinoMed,PubMed,EMBASE and Cochrane were searched with the keywords of Lyciurn Barbarurn,cirrhosis and liver fibrosis.Then,the obtained literature was screened,and the basic data and effect size of each study,including the overall symptoms,liver enzymogram(ALT,AST),total bilirubin and liver fibrosis markers(hyaluronic acid,laminin and type Ⅲ procollagen)were extracted.After evaluating the quality of the included literature with reviews manager 5.3,the overall treatment effect,liver function and liver fibrosis indexes was acessed.Among them,the dichotomous variables were analyzed with risk ritio(95%confidence interval),and the continuous variables were analyzed with standard mean deviation.The heterogeneity of the included studies was judged by I2,and P<0.05 was considered to be statistically different.Results:a total of 10 clinical studies were included.Results showed that,Lycium barbarum contained Traditional Chinese Medicine could improve the overall symptoms(P<0.00001,RR=1.27,95%CI[1.16,1.38],I2=0%),alanine aminotransferase(P=0.02,SMD=-0.68,95%Cl[-1.24,-0.13],I2=83%)and total bilirubin(P<0.0001,SMD=-0.81,95%CI[-1.20,-0.42],I2=93%)also decreased significantly compared with the conventional treatment.Further more,liver fibrosis markers,including hyaluronic acid(P=0.0004,SMD=-0.93,95%CI[-1.44,-0.42],I2=23%),laminin(P=0.0004,SMD=-0.90,95%CI[-1.40,-0.40],I2=0%),type Ⅲ procollagen(P=0.005,SMD=-1.27,95%CI[-2.15,-0.39],I2=57%)were significantly improved than conventional treatment.Conclusions:Lycium Barbarum contained Traditional Chinese Medicine can improve the symptoms of liver cirrhosis.Lycium Barbarum can be added for the treatment of liver cirrhosis.Part Ⅱ DNA methylation and gene expression in hepatic cirrhosisObjectives:to identify differentially expressed genes and differentially methylated genes in liver fibrosis,and to explore the relationship between gene methylation and gene expression.Find out the important genes and pathways with methylation changes in liver fibrosis.Methods:R studio software and the related R packages were used to screen the differentially expressed genes(DEGs)and differentially methylated genes(DMGs)in liver cirrhosis,and showing the results in heatmaps and figures.The clusterprofiler package and other online tools were used to analyze the pathways involved DMGs.Results:a total of 357 DEGs and 8830 DMGs were identified.The cross analysis shows that 411 gene were positively correlated with their DNA methylation,while 235 gene were negatively correlated with their DNA methylation,and the expression of other DMGs genes remain unchanged.The pathway enrichment analysis of DMGs identified several important pathways with methylation varation,such as PI3K Akt,MAPK,cGMP PKG,JAK-STAT,TGF beta,NF-κB,and PPAR,etc.Conclusions:there is no clear correlation between gene methylation and gene expression,and NF-κB was screened for follow-up study.Part Ⅲ The function and mechanism of betaine in hepatic fibrosisObjectives:to study the effect of betaine on hepatic fibrosis and its mechanism.Therefore,we studied the inhibitory effect of betaine on the differentiation of hepatic stellate cells in cells and the inhibitory effect of betaine on fibrosis in vivo.Methods:betaine was studied in HSC-T6 cells and rats.Firstly,CCK-8 assay and molecular analysis were used to explore the effect of betaine on the fibroblastic ability of HSC-T6 cells.Secondly,in the rat model induced by carbon tetrachloride,the liver fibrosis was assessed with various staining,and NF-κB and Dnmt3a wwere detected by immunoblotting and immunohistochemistry.Finally,NGS sequencing was used to determine the methylation status of the second CpG island of RelA,which is the subunit of NF-κB.Results:the application of high-dose betaine did not inhibit cell activation in HSC-T6 cells.The expression of α-SMA、colⅣ α1 and fibronectin was reversed by betaine.In addition,extracellular matrix deposition was reversed by betaine in rat liver.Downregulation of p65 and p50 in HSC-T6 cells and rat liver by betaine indicates that betaine alleviating liver fibrosis through NF-κB signal pathyway.The expression of three DNA methyltransferases decreased simultaneously in HSC-T6 cells and rat liver.Interestingly,the decreasing of p65 level is accompanied by the decreasing of DNMT family,which indicates that there may be a correlation between the two genes.NGS sequencing showed that the methylation status of-180 cytosine at the second CpG island in the promoter of RelA decreased,but other cytosines shows no significant change.Conclusions:the inhibitory effect of betaine on HSCs activation and liver fibrosis have been confirmed in vivo and in vitro.And betaine may relieve liver fibrosis through hypomethylation of-180 cytosine of RelA gene sequences. |
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